Sukesh K. Gupta,
Swetlana Gautam,
Jitendra K. Rawat,
Manjari Singh,
Shubhini A. Saraf and
Gaurav Kaithwas*
Department of Pharmaceutical Sciences, School of Biosciences and Biotechnology, Babasaheb Bhimrao Ambedkar University, Vidya Vihar, Raebareli Road, Lucknow 226 025, India. E-mail: gauravpharm@gmail.com; gauravpharm@hotmail.com; Tel: +91-522-2998129, +91-9670204349
First published on 18th December 2014
The aim of the present study was to study in detail the effect of variable doses of aspirin on intestinal toxicity. Albino rats were randomly divided into six groups and subjected to 13 weeks treatment against a sham control (3 ml kg−1 by mouth (p.o.) normal saline); a toxic control (2.5 ml kg−1 intraperitoneal injection (i.p.), MTX); a low dose of aspirin (8 mg kg−1, p.o.); a low dose of aspirin plus MTX (8 mg kg−1, p.o. + 2.5 ml kg−1, i.p.), a high dose of aspirin (45 mg kg−1, p.o.); and a high dose of aspirin plus MTX (45 mg kg−1, p.o. + 2.5 ml kg−1, i.p.). The intestinal toxicity of aspirin was assessed on the basis of biochemical changes and modulation in the inflammatory markers. Low doses of aspirin gave significant protection against MTX-induced toxicity, whereas high dose failed to do so. High doses of aspirin also produced adverse biochemical changes in the physiology, but low dose did not.
MTX-associated chemotherapy may directly induce mucositis by causing deoxy-ribonucleic acid (DNA) strand breakage, either by generating ROS or by provoking enzymatic or transcription factor in cellular elements within the mucosa. ROS may damage cells other than cancerous tissues and may also activate secondary mediators of injury, including transcription factors such as nuclear factor-κβ (NF-κβ).5 Activation of transcription factors in response to ROS, radiotherapy or chemotherapy results in gene up-regulation for tumor necrosis factor-α (TNF-α) and interleukins (IL-1β and IL-6).6 Previous studies have investigated changes in levels of NF-κβ and other pro-inflammatory cytokines, including tumor necrosis factor (TNF)-α, and also interleukin (IL)-1β and IL-6 in experimental MTX-treated animals.7,8 This leads to tissue injury and apoptosis of cells in the submucosa and primary injury of cells within the basal epithelium, and also to mucositis.9 MTX-induced injury within the intestinal tissue is thus associated with hyperpolarization, and injury to the mucosal, submucosal and basal epithelium due to ROS production, and consequent regulation of pro-inflammatory cytokines and mediators.6
MTX is often used as an initial disease-controlling antirheumatic agent by modifying the immune system, and is commonly prescribed with other drugs, including non-steroidal anti-inflammatories (NSAIDs), like aspirin.10 In the past, there have been safety concerns about the use of painkillers, NSAIDs and aspirin, with MTX in patients with inflammatory disorders.11 The simultaneous use of these drugs with MTX may increase the risk of toxicity, causing mouth ulcers, nausea, vomiting, hepatotoxicity, cardiotoxicity or bone marrow depression.12,13 In these cases, close monitoring of the vital parameters is essential.
In view of the therapeutic advantages of the MTX– and NSAID–aspirin combination, and the risk of associated toxicities, it was considered to be worth assessing the effect of low and high doses of aspirin accompanying the administration of MTX.
S no. | Group | Treatment | pH | Total acidity (mEq l−1) | Free acidity (mEq l−1) | CMDI |
---|---|---|---|---|---|---|
a Each group contained six animals. Values are represented as mean ± SD.b Statistical significance was compared to toxic control using one-way ANOVA followed by Bonferroni test (*p < 0.05, **p < 0.01 and ***p < 0.001).c Values in parenthesis represent percentage inhibition. | ||||||
1. | Group I | Sham control (normal saline, 3.0 ml kg−1, p.o.) | 7.15 ± 0.02 | 10.99 ± 1.67 | 7.32 ± 1.96 | 0.17 ± 0.40 |
2. | Group II | Toxic control (MTX, 2.5 ml kg−1, i.p.) | 6.27 ± 0.04 | 14.38 ± 1.42 | 11.92 ± 0.92 | 4.00 ± 0.00 |
3. | Group III | Aspirin (8 mg kg−1, p.o.) | 7.67 ± 0.02*** | 10.52 ± 0.45*** (26.84%) | 7.94 ± 0.45*** (33.38%) | 0.83 ± 0.40*** (79.25%) |
4. | Group IV | Aspirin + MTX (8 mg kg−1, p.o. + 2.5 ml kg−1, i.p.) | 7.40 ± 0.02*** | 10.52 ± 0.43*** (26.84%) | 7.70 ± 0.51*** (35.40%) | 1.83 ± 0.40*** (54.25%) |
5. | Group V | Aspirin (45 mg kg−1, p.o.) | 7.73 ± 0.06*** | 11.37 ± 0.64*** (20.93%) | 8.88 ± 0.58*** (25.50%) | 1.83 ± 0.40*** (54.25%) |
6. | Group VI | Aspirin + MTX (45 mg kg−1, p.o. + 2.5 ml kg−1, i.p.) | 7.50 ± 0.02*** | 14.47 ± 0.77 (0.76%) | 11.40 ± 0.77 (4.36%) | 2.83 ± 0.75** (29.25%) |
S no. | Group | TBARS (nm of MDA per mg of protein) | GSH (mg %, 1 × 10−4) | SOD (unit of SOD per mg of protein) | Catalase (nm at H2O2 per min per mg of protein) | Protein carbonyl (nm ml−1) |
---|---|---|---|---|---|---|
a Each group contained six animals. Values are represented as mean ± standard deviation.b Statistical significance is compared to toxic control using one-way ANOVA followed by Bonferroni test. *p < 0.05, **p < 0.01, and ***p < 0.001 were considered statistically significant. | ||||||
1 | Group I | 0.71 ± 0.04 | 22.40 ± 0.55 | 16.47 ± 4.47 | 7.70 ± 0.81 | 50.45 ± 0.39 |
2 | Group II | 2.91 ± 0.02 | 113.32 ± 0.01 | 42.95 ± 6.93 | 37.46 ± 1.60 | 116.81 ± 0.39 |
3 | Group III | 0.79 ± 0.01** | 26.7 ± 2.44** | 26.73 ± 6.89** | 8.57 ± 1.01*** | 70.40 ± 1.55*** |
4 | Group IV | 1.35 ± 0.01*** | 61.8 ± 3.13*** | 48.74 ± 8.14*** | 23.07 ± 2.27*** | 104.47 ± 1.35*** |
5 | Group V | 1.25 ± 0.01*** | 28.2 ± 2.82*** | 37.21 ± 7.23*** | 31.11 ± 2.91*** | 94.76 ± 3.92*** |
6 | Group VI | 1.47 ± 0.02*** | 98.7 ± 4.79*** | 44.52 ± 9.50*** | 34.62 ± 7.30*** | 109.50 ± 2.00* |
The intestinal tissues were examined for the presence of pro-inflammatory (IL-2) and anti-inflammatory cytokines (IL-4 and IL-10), and these were seen to be increased after MTX treatment. The administration of aspirin gave an additional increase (Table 3).
S no. | Group | IL-2 (pg ml−1) | IL-4 (pg ml−1) | IL-10 (pg ml−1) |
---|---|---|---|---|
a Each group contained six animals. Values are represented as mean ± standard deviation.b Statistical significance is compared to toxic control using one-way ANOVA followed by Bonferroni test: compare all pairs of columns; *p < 0.05, **p < 0.01, ***p < 0.001 were considered statistically significant. | ||||
1 | Group I | 524.80 ± 0.00 | 108.05 ± 5.19 | 82.66 ± 5.95 |
2 | Group II | 764.82 ± 115.94 | 238.86 ± 46.84 | 876.98 ± 213.07 |
3 | Group III | 526.99 ± 156.71 | 257.31 ± 41.94 | 177.82 ± 0.00*** |
4 | Group IV | 968.17 ± 133.23** | 300.08 ± 49.92** | 1167.60 ± 79* |
5 | Group V | 483.01 ± 31.46* | 284.28 ± 15.34 | 1699.10 ± 66.96** |
6 | Group VI | 683.66 ± 70.41 | 223.87 ± 0.00 | 224.91 ± 25.92** |
In view of the cardiac and hepatotoxicity associated with MTX treatment, the cardiac and liver enzymatic markers were also assayed biochemically. The administration of MTX generated significant toxicity in the cardiac tissue, clearly evident from increased MDA generation (0.93 ± 0.01) along with raised enzymatic activity of the LDH (323.01 ± 17.17), catalase (22.02 ± 3.16), and SOD (56.77 ± 2.92) against the sham control. Administration of low dose aspirin produced some restoration of MDA generation and LDH activity. An insignificant reduction in the enzymatic activities of catalase and SOD was seen after aspirin treatment (Table 4).
S no. | Group | LDH (unit per litre) | TBARS (nm of MDA per mg of protein) | Catalase (nm at H2O2 per min per mg of protein) | SOD (unit of SOD per mg of protein) |
---|---|---|---|---|---|
a Each group contained six animals. Values are represented as mean ± standard deviation.b Statistical significance compared to toxic control using one-way ANOVA followed by Bonferroni test; *p < 0.05, **p < 0.01, ***p < 0.001 were considered statistically significant. | |||||
1 | Group I | 226.66 ± 11.44 | 0.48 ± 0.03 | 5.27 ± 0.36 | 19.16 ± 8.67 |
2 | Group II | 311.66 ± 17.17 | 0.93 ± 0.01 | 22.02 ± 3.16 | 56.77 ± 2.92 |
3 | Group III | 239.80 ± 4.24* | 0.55 ± 0.01** | 4.84 ± 0.51** | 25.92 ± 0.41*** |
4 | Group IV | 307.21 ± 0.57** | 0.79 ± 0.02*** | 19.46 ± 2.41 | 53.98 ± 2.21 |
5 | Group V | 266.85 ± 4.97 | 0.62 ± 0.01** | 7.16 ± 4.02*** | 33.93 ± 12.11*** |
6 | Group VI | 403.30 ± 9.67** | 0.86 ± 0.03** | 29.75 ± 10.07 | 56.87 ± 2.76 |
When assessed on the basis of the hepatic enzymes, MTX was found to be hepatotoxic, with increased levels of SGOT (44.50 ± 1.95) and SGPT (43.96 ± 0.88) in comparison to the control. Treatment with low doses of aspirin allowed an increased level of SGOT, and successively higher doses were seen to further increase enzymatic levels. In addition, aspirin at both low and high dosage also increased the SGPT levels (Table 5). Scanning electron microscopy of the intestinal tissue in the control group gave the clear impression of an overall villous and mucous pattern, and treatment with MTX produced hyper-polarization, loss of mucus and a distorted villous structure (Fig. 1).
S. no. | Group | SGOT (unit dl−1) | SGPT (unit dl−1) |
---|---|---|---|
a Each group contains six animals. Values are represented as mean ± SD.b Statistical significance compared to toxic control using one-way ANOVA followed by Bonferroni test: compare all pairs of columns,*p < 0.05, **p < 0.01, and ***p < 0.001 were considered statistically significant. | |||
1 | Group-I | 23.34 ± 0.20 | 20.88 ± 1.51 |
2 | Group-II | 44.50 ± 1.95 | 43.96 ± 0.88 |
3 | Group-III | 24.69 ± 1.01*** | 30.49 ± 0.64** |
4 | Group-IV | 30.38 ± 1.25** | 52.65 ± 0.98** |
5 | Group-V | 40.62 ± 0.66** | 36.21 ± 3.77** |
6 | Group-VI | 58.70 ± 1.80*** | 65.73 ± 1.79*** |
Treatment with low doses of aspirin was seen to give protection against intestinal toxicity when observed through physiological parameters. The aspirin treatment gave a significant increase in intestinal pH, with a decrease in both total and free acidity. The CMDI score in the toxic control was also significantly reduced by aspirin in a dose-dependent manner; indeed aspirin gave marked protection against MTX-induced intestinal toxicity.
Diversification in the antioxidant defense mechanisms in the intestinal tissue associated with MTX treatment has been extensively reported and is an accepted mechanism of MTX toxicity. The study in fact showed increased MDA production in the MTX-treated group.
MDA is a product of phospholipid peroxidation and is a marker for oxidative stress. The regimen with aspirin reduced MDA production to a reasonable level. It was significant that an antiplatelet (low) dose of aspirin was more appropriate than an anti-inflammatory (high) dose.
Glutathione (GSH) plays a dominant role in insulating tissues from peroxidative attack, and MTX significantly increased the level of tissue GSH, contradicting earlier findings suggesting depletion of GSH in tissues during oxidative stress.28 The increased GSH levels observed in our study might possibly have been the result of increased biogenesis of GSH due to oxidative stress, the product of a feedback mechanism. The administration of aspirin with MTX gave a significant decrease in tissue GSH levels, and this could have been due either to the restored biogenesis of GSH or to diminished levels of oxidative stress as a result of aspirin therapy.29,30
The combined effect of SOD and catalase provides a major defense against ROS. SOD forms hydrogen peroxide through its scavenging action on the superoxide radical, and this scavenging effect is further assisted by catalase (a haem protein), which catalyzes the breakdown of hydrogen peroxide to H2O and molecular oxygen, thus protecting the tissue from highly reactive OH− radicals. We observed increased enzymatic activity of SOD and catalase, in disagreement with previous studies, which have mostly observed for decreased levels of SOD, and subsequently catalase, as a consequence of oxidative stress.31 In this study, we were open also to the possibility of increased activity of both SOD and catalase. One could foresee a diminished activity of SOD and catalase as a result of an amelioration of oxidative stress, but we considered that physiological compensatory mechanisms for dealing with oxidative stress could be a possible reason for our observations.
A simultaneous increase in catalase activity is essential in order to outweigh the superoxide scavenging caused by SOD, and this was observed in our study. ROS can damage all types of biological molecules, in addition to lipids, DNA and proteins. The alterations to proteins directly caused by oxidative stress on amino acid residues can be linked to the disposition of carbonyl derivatives, a widely used marker for protein oxidation.32 Studying the formation of protein carbonyls has an advantage over lipid peroxidation, as oxidized proteins are more substantial and the protein carbonyl is therefore universally selected as a marker of oxidative stress.23 In the present study, MTX produced a noticeable increase in protein carbonyl content, denoting protein oxidation. Aspirin at antiplatelet dose level produced a substantial decrease in protein carbonyl content in comparison to MTX-treated groups, and aspirin normalised the levels of physiological antioxidant defence. The antiplatelet dose of aspirin produced a more effective response towards MTX toxicity than a high dose.
To give further insight, we examined the role of pro- and anti-inflammatory cytokines in intestinal tissues, and found a significant increase in IL-2, IL-4 and IL-10 levels following administration of MTX.
IL-2 is a signalling molecule in immune systems and oversees the activities of the leukocytes and lymphocytes responsible for immunity. An increased level of IL-2 expression was revealed after MTX treatment, and in addition aspirin (low dose) increased the level of IL-2 when administered in conjunction with MTX. On the other hand, a higher dose of aspirin produced an insignificant decrease in the IL-2 level, due to the simple fact that this was an anti-inflammatory dose. This finding did not agree with previous studies, which found decreased levels of IL-2 in the serum of patients, including rheumatoid patients, treated with MTX.33
A previous study has shown that ex vivo treatment of peripheral blood monocytes with MTX increased the expression of IL-4 and IL-10, which is in line with the present study. However, aspirin did not produce any lowering of IL-4 levels, either at high or low dose. IL-10 is an anti-inflammatory cytokine and underwent a striking increase following the MTX treatment, adopting an immuno-compromised status due to its immuno-regulatory properties. A high dose of aspirin significantly reduced the levels of IL-10, whereas the low dose produced increased IL-10 levels. In addition to the other dominant toxicities, MTX has been noted both clinically and preclinically to be cardiotoxic and hepatotoxic.34 Taking this into account and to gain a better understanding, we looked at serum SGOT and SGPT levels. The results demonstrated greatly increased hepatotoxicity, accompanied by a large increase in serum SGOT and SGPT levels. Treatment with low doses of aspirin produced considerable protection by normalizing the serum SGOT and SGPT levels. Similarly, cardiac toxicity was conspicuous through increased enzymatic activity of LDH, SOD and catalase, along with noticeable embodiment of MDA products after MTX treatment. Aspirin, at low doses, gave protection against MTX-induced cardiotoxicity. This could be credited to blockage of the COX enzyme through the acetylation of serine-529 residue. The COX-1 inhibition in the platelets leads to inhibition of TXA2 production, which is a key platelet aggregator. Moreover, in endothelial cells COX-1 accelerates the formation of prostacyclin, which is a vital vasodilator. The dynamic balance of TXA2 and prostacyclin modulated by aspirin provides the required balance.35
When observed microscopically, the administration of MTX demonstrated significant hyperproliferation, loss of mucus and distorted villous structure, which is in concordance with earlier reports.16,36 In comparison to MTX or aspirin (high dose), a low dose of aspirin gave significant protection either alone or in combination. The results of microscopy in the present investigation suggested that a low aspirin dose offered an advantage over high doses in combating intestinal toxicity induced by MTX.
From the foregoing investigation, one can deduce that a low dose of aspirin can provide a significant counter against MTX-induced intestinal toxicity, cardiotoxicity and hepatotoxicity. The quoted effect of low dosage aspirin could be due to its antiplatelet action, leading to impaired production of thromboxane, which binds with platelets to form a patch over damaged blood vessels. The patch may eventually become too large and block the blood flow, both locally and downstream, which is nowadays implicated against the clinical use of aspirin.37 On the contrary, a high dose can advance the toxicity; in fact a low dose of aspirin can be considered a complementary therapy in MTX treatment. The use of aspirin with MTX may offer advantages, as the renal clearance of MTX or its metabolite 7-hydroxyl-MTX is not clinically hindered by aspirin.38,39 In future, the combination of a low dose of aspirin with MTX could offer a better pharmacological profile, with less toxicity. However, the use of aspirin in combination with MTX in the clinical management of rheumatoid arthritis (RA) is in question and needs to be fully investigated.
From the above evidences, we can conclude that a low dose of aspirin can be considered as an adjuvant therapy with MTX chemotherapy and other treatment regimens, which may then reduce toxicity. However, the suitability of MTX and aspirin in the clinical management of RA is open to question. Further studies will be needed to confirm the appropriateness of the use of aspirin as an adjuvant to MTX in the clinical sphere.
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